Detection and structural features of the βB2-B3-crystallin heterodimer by radical probe mass spectrometry (RP-MS)

The predilection of the beta-crystallin B2 subunit to interact with the beta B3 subunit rather than self associate is evident by the detection of the beta B2-B3-crystallin heterodimer by native gel electrophoresis and electrospray ionisation time-of-flight (ESI-TOF) mass spectrometry under non denaturing conditions. The complex has been detected for the first time and its molecular mass is measured to be 47 450 +/- 1 Da. Radical probe mass spectrometry (RP-MS) was subsequently applied to investigate the nature of the heterodimer through the limited oxidation of the subunits in the complex. Two peptide segments of the beta B2 subunit and six of the beta B3 subunit were found to oxidise, with far greater oxidation observed within the beta B3 versus the beta B2 subunit. This, and the observation that the oxidation data of beta B2 subunit is inconsistent with the structure of the beta B2 monomer, demonstrates that the protection of beta B2 is conferred by its association with beta B3 subunit within the heterodimer where only the residues of, and towards, its N-terminal domain remain exposed to solvent. The results suggest that the beta B2 subunit adopts a more compacted form than in its monomeric form in order for much of its structure to be enveloped by the beta B3 subunit within the heterodimer. Copyright (C) 2009 John Wiley & Sons, Ltd.